Fig 2: Xenopus K19 associates with 14-3-3 proteins and C-cadherin. (A) Pan 14-3-3 immunoprecipitates (1% Tergitol type NP-40) from whole embryo lysates prior to band extraction and processing using LC/MS-MS. Prominent bands at 48, 30, and 28 kDa were processed. Heavy chain IgG from the antibody used for IP was not excised. (B) Table summary of relevant proteins detected in gel extracts processed using LC/MS-MS. Experiments were conducted using 14-3-3 immunoprecipitates from whole embryo lysates (WEL) as well as lysates from mesendoderm (ME) tissue only. Analysis was performed using Scaffold 4.7.3. (C) Summary schematic of K19 peptides (red) detected in the 48 kDa sample. Peptides are depicted within the context of the K19 primary structure and alongside described (green) and predicted (blue) possible 14-3-3 interaction sites. (D) 14-3-3 proteins were immunoprecipitated (1% Tergitol type NP-40) from whole embryo lysates and immunoblotted for C-cadherin, K19, and Vinculin. C-cadherin band is denoted by an arrow. The bottom band is yolk protein from sample.

Fig 3: 14-3-3 is necessary for targeting of keratin to cell–cell contacts. (A–C) Mesendoderm cell pair establishing de novo cell–cell contact after collision. Cells are expressing mCherry-R18M and eGFP-K19. The arrowhead depicts the cell–cell adhesion where keratin densities (asterisk) have localized. Image stack is 35 slices (10.54 µm). (D–F) Postcollision mesendoderm cell pair expressing mCherry-R18 and eGFP-K19. The bracket depicts the cell–cell adhesion that demonstrates a gap where keratin filaments have failed to reorganize. Image stack is 40 slices (10.92 µm). (G) Comparison of the number of keratin gaps at cell–cell adhesions in postcollision cell pairs expressing either mCherry-R18M or mCherry-R18. Analysis was performed using a z-test for proportions from two samples, ***p < 0.001. Error bars represent the 99.9% CI for each sample proportion. Fluorescent images are z-stacks (maximum intensity projection). Brightfield images are single planes. Scale bars are 20 µm.

Fig 4: 14-3-3 proteins target keratins to cell–cell adhesions. (A) Schematic of fusion peptides created by the insertion of R18 or R18M (R18/M) into FLAG/eGFP-K19. This construct was generated using full-length K19.L from X. laevis. (B) Protein lysates from stage 10.5 Xenopus embryos expressing C-cadherin-eGFP and either FLAG-R18-K19 or FLAG-R18M-K19 were prepared in 1% Triton X-100. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (C) Protein lysates from stage 10.5 Xenopus embryos expressing human FLAG-14-3-3ß and either eGFP-R18-K19 or eGFP-R18M-K19 were prepared in 1% Tergitol-type NP-40. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (D, E) Explanted mesendoderm cells mosaically expressing eGFP-R18M-K19 (in D) or eGFP-R18-K19 (in E). (F, G) Explanted mesendoderm coexpressing mem-RFP and eGFP-R18M-K19 (in F) or eGFP-R18-K19 (in G). Arrows indicate areas where filament densities have localized. Images are z-stacks (maximum intensity projection). Scale bars are 10 µm.

Fig 5: Generation of the salivary gland organoid in 3D ESC cultures. a Culture protocol (top) and schematic diagram (bottom) of salivary gland differentiation from ESCs. ESCs embryonic stem cells, ME mesendoderm, DE definitive ectoderm, NE neural ectoderm, nne non-neural ectoderm, EPI epidermis, OE oral ectoderm, OE-SG salivary gland placode, SG salivary gland. b Phase-contrast representative images of aggregates on day 1 and 8 (left and middle). pan-cytokeratin (Pan-CK, red) and Sox9 (green) were detected via immunofluorescence analysis (right). Scale bars, 300 µm. c Real-time RT-PCR analysis of oral ectoderm genes in the aggregates on day 8 with or without growth factors and inhibitors and in the head at E13.5. This experiment was replicated three times with similar results. The results are presented as the mean ± S.D. and were normalized to GAPDH. Statistical analyses were performed using Student’s t-test; *P = 0.0000009, **P = 0.014. d, e The outer layer corresponding to the oral epithelium was resected after infection with Sox9 and Foxc1 adenoviruses and cultured. Schematic representation (d) and phase-contrast representative image of the resected outer layer at d8 and the culture at d15. Scale bars, 300 µm (e). f Phase-contrast representative images showing the morphological changes in the aggregates at d23. The yellow square outline indicates epithelial bud formation (left). Scale bars, 300 µm (left), 50 µm (right). g Bright-field view of the aggregate at d28. Arrowheads indicate the epithelial branching structure. Scale bar, 300 µm. h, i Immunofluorescence images of the aggregates at d23 and d28 (h) and mouse embryonic SMG at E15.5 and E18.5 (i). The epithelial marker Pan-CK (green), the ductal marker K18 (green), the acinar cell marker AQP5 (red), and the myoepithelial cell marker a-SMA (red) were detected. Representative images from one out of more than three experiments are shown. Scale bars, 50 µm. Arrowheads indicate an epithelial bud (E). Arrows indicate ducts (D)